CONTRIBUnON TO THE LIFE HISTORY 

AND PHYSIOLOGY OF CYLINDRO- 

SPORIUM ON STONE FRUITS 



A THESIS 

Presenti':d to thi': Faculty of the Graduate School 

OF Cornell University for the dilgree of 

DOCTOR OF PHILOSOPHY 



BY 



BASCOMBE BRITT HIGGINS 



ReiM-inted from American Journal of Botany, I: i4S-i73- April, 1914- 



CONTRIBUTION TO THE LIFE HISTORY 

AND PHYSIOLOGY OF CYLINDRO- 

SPORIUM ON STONE FRUITS 



A THESIS 

Presented to the Faculty of the Graduate School 

OF Cornell University for the degree of 

DOCTOR OF PHILOSOPHY 



BY 

BASCOMBE BRITT HIGGINS 



Reprinted from American Journal of Botany, 1: 145-173. April, 1914. 






JUL 



9 '--^ 



[Reprinted from American Journal of Botany, Vol. I, No. 4. April, 1914.I 



CONTRIBUTION TO THE LIFE HISTORY AND 

PHYSIOLOGY OF CYLINDROSPORIUM ON 

STONE FRUITS^ 

Bascombe Britt Higgins 

with elates xiii-xvi 

Introduction. 

The disease of plums and cherries caused by the fungus CyHndro- 
sporium has long been known, and because of its economic importance 
and peculiar appearance has for many years attracted the attention 
of horticulturists and plant pathologists both in Europe and America. 
On certain species of the hosts the disease becomes very conspicuous 
because of the "shot hole" efifect on the leaves, produced by the 
dropping out of roundish areas of diseased tissue. From the leaves 
of other species however the spots do not drop out, and usually in such 
cases the leaf tissue is not killed to any appreciable extent. In cases 
of severe attack the leaves often turn yellow and drop prematurely, 
which of course interferes more or less seriously with food production 
and the future welfare of the tree. 

"Shot holes" are produced in the leaves of stone fruits by several 
different organisms or even by mechanical injury, for example, as the 
result of a needle prick. Duggar (8) found also that they were pro- 
duced readily by spraying plants with poisonous solutions. However, 
the fungus Cylindrosporium is probably the most prolific cause of 
this phenomenon, at least in cherries; and many collectors have 
apparently attributed all "shot hole" effects on species of Prunus to 
Cylindrosporium padi Karst. 

1 Contribution from the Department of Botany, Cornell University. No. 155. 

[The Journal for March (1: 97-144) was issued 6 May 19 14.] 

145 



146 BASCOMBE BRITT HIGGINS 

This fungus was described by Karsten (17) in 1884 from leaves 
of Prumis padus; and Sorauer (13) states that in Europe the disease 
is confined almost entirely to this species. Aderholdt (i) in 1901 
said that the disease had been common on both sweet and sour cherries 
during the previous ten years. In America a disease attributed to 
this prganism has been reported on nearly all species of Prunus both 
wild and cultivated. 

Because of the prevalence of the disease and its consequent econ- 
omic importance, it has seemed very desirable to know the complete 
life history of the fungus causing it. With this purpose in view an 
investigation has been in progress during the past three years, the 
results of which are here reported. 

For several years the disease has been very abundant in the 
vicinity of Ithaca, Ngw York, on the sweet cherry (P. avium) and the 
wild choke cherry (P. virginiana). It has been found less abundant 
on the sour cherry (P. cerasus, P. mahaleb, P. pennsylvanica) , and on 
the plums (P. domestica, P. insititia, and P. spinosa). Through the 
courtesy of Professor J. G. Hall (now of Pullman, Washington) leaves 
of P. serotma affected with the disease were obtained from Clemson 
College, South Carolina, in August, 1912. A Septoria having spores 
only slightly different, but produced in a pycnidium, also found on P. 
pennsylvanica, was studied for comparison with these. The results 
of this study of Septoria will be reported in another paper. 

This abundance of material on a number of host species has made 
possible a comparative study of structural characters, relation to the 
host tissue, cultural characters, interrelations of the fungus on the 
different hosts, and its life history. Pure cultures of the organism 
from P. spinosa were not obtained, so that it could not be included in 
all the comparisons. 

Structural Characters 

The genus Cylindrosporium is characterized by having elongated 
colorless conidia borne on a more or less disk-shaped stroma just 
beneath the host epidermis. The acervuli on all the species of Prunus 
under observation agree with this characterization. The stroma is 
very delicate, at first consisting of one cell layer only, but becomes 
slightly thicker as the acervulus grows older. On the outer surface 
of this stroma short conidiophores give rise to conidia. They develop 
first over the center of the stroma and continue to develop centri- 



CYLINDROSPORIUM ON STONE FRUITS I47 

fugally as the latter grows in diameter. This growth of the stroma is 
lateral, extending out between the epidermis and mesophyll of the 
leaf, and never turns up at the edges so as to resemble a pycnidial 
structure.2 The stroma which bears conidia lies under the epidermis 
on either surface of the leaf. When the conidia have accumulated 
in sufficient number the epidermis is broken and they appear usually 
as a whitish or yellowish white mass above the stroma. In P. serotina 
(more rarely in other species) the stiff cuticle prevents the formation 
of a large opening and the conidia are forced out in long tendrils.^ 

The conidia from all the host species are very similar. They are 
long, slender, curved or flexuous, and continuous or 1-3 septate.* 
There are a few minor differences which are not very distinct, since 
the conidia vary considerably on each host. The conidia from plum 
leaves (all three species) were more constant than those from cherry 
leaves. Here they are blunt at the proximal end and taper gradually 
toward the apex, and they are mostly once septate. The largest spores 
found were from P. serotina and P. virginiana. On both species spores 
measuring 45-80^ in length were found. On no other species were 
they found as long as So/j.. 

^ Septoria cerasina Peck (29th Report N. Y. State Mus. Nat. Hist. 48, 1878) 
the type material of which, through the kindness of Dr. Peck. I have had an oppor- 
tunit}' of examining, has a typical fruiting structure of this description (fig. 4). 
The same is true of the specimen distributed in "Fungi Americana" No. 747 as 
Septoria Ravenellii Thum, which is apparently identical with S. cerasina Peck. 
This is not the case, however, with Septoria pruni Ellis, which Aderholdt (i) includes 
as a synomym of Cylindrosporium padi Karst. Here a distinct pycnidial structure 
surrounds the spores which shows it to be a true Septoria. This was not from type 
material, but from the specimen in Ellis' North America Fungi 1151 in the Cornell 
University herbarium. It is from the same collection which Aderholdt must have 
examined, though of course in such a large collection some leaves affected with 
Cylindrosporium might also be included. 

^ This accounts for Peck's (23) statement that 5. cerasina differs from Cylindro- 
sporium padi (on P. domestica) in this character. 

* Aderholdt (i) says that the conidia are not truly septate but have false cross 
walls. He does not state how this was demonstrated. Pammel (22) says that the 
cross walls react to stains in exactly the same way that the side walls react, and this 
has been confirmed by my own studies. Haidenhain's iron alum haematoxylin 
Delafield's haematoxylin, and Flemming's triple stain all bring out the cross walls 
very distinctly. Very often also the cells become constricted at the cross walls 
upon germination; or the contents of one cell may be lost entirely without appar- 
ently affecting the rest of the conidium. 



148 bascombe britt higgins 

Cultural Characters 

The conidia seem to lose their vitality very rapidly on drying. 
When they are taken from the dry masses which cover the acervuli 
only a small per cent of them germinate in nutrient agar or in tap 
water; and some taken from cherry leaves which had not been wet 
for about a month failed to produce infection on other leaves of the 
same plants. 

Fresh spores germinate very slowly in agar. For this reason and 
because of the large number which failed to germinate it was found 
very difficult to obtain pure cultures of the fungus by the dilution 
method. When leaves were placed in a moist chamber for a few hours 
so that fresh conidia were formed, pure cultures were readily obtained 
by picking up a quantity of them on the point of a needle and then 
dragging the needle across an agar plate. After four or five days 
there were usually spots on some of the streaks free from bacteria and 
other fungi where some of the Cylindrosporium conidia were beginning 
to germinate. Blocks of agar containing such germinating conidia 
were then transferred to tubes of agar or to sterilized bean pods. 

Comparison of the fungus isolated from the different hosts was 
made chiefly from growth on steamed bean pods; but the nature of 
the medium seemed to have very little effect on the nature of the 
growth, colonies very similar to those on bean pods being produced 
on agar dissolved in tap water; on agar to which had been added an 
extract of beans, potatoes, malt extract, prunes, or cherry leaves; 
on sterile slices of pear; or on steamed cherry leaves. 

Growth from the spores is always very slow, and is usually not 
apparent to the unaided eye until 10-15 days have passed. At this 
time it appears as a small whitish speck, which when examined 
microscopically is found to consist chiefly of a stroma covered with 
quantities of conidia similar to those produced on the host plant. 
This stroma grows slowly, enlarging until a hemispherical mass 0.5-1 
cm. in diameter is formed, which occurs in about two months. Before 
this time the stroma has turned coal black and has a carbonaceous, 
crust-like appearance in the fungus isolated from P. domestica and P. 
insititia. After the stroma has turned black few spores are produced 
even when transferred to new media. The stroma produced from the 
fungus isolated from P. avium, P. cerasus, and P. pennsylvanica some- 
times turns dark but never black and crust-like as in that from the 
plums. The stroma from the other three cherries, P. virginiana, P. 



CYLINDROSPORIUM ON STONE FRUITS I49 

serotina, and P. mahaleh, has never been observed to turn black and 
has usually a creamy white and more floccose appearance. 

Relation between Fungus and Host 

Interest in the physiological relation between the fungus and the 
host tissue was aroused by the observation, before mentioned, that 
the spots containing Cylindrosporium are deciduous in some species 
while in others they are persistent. Even in the same species there 
is marked variation in this respect. This variation is very striking 
in P. virginiana where ragged remnants of leaves, from which dozens 
of spots have dropped out, may later be abundantly infected with 
apparently little injury to the leaf tissue except the killing of a few 
cells in immediate contact with the acervulus. Because of this marked 
variation as host, P. virginiana was used chiefly in this part of the 
investigation which was undertaken in the hope of finding some 
explanation for this phenomenon. 

The first problem was to find, by a histological study, what occurs 
just before and at the time of the dropping of the spots. For this 
purpose a series of spots were cut out so as to include some of the sur- 
rounding healthy tissue. These were killed, embedded in parafifin, 
and sectioned. The series began with the first sign of infection (a 
slight yellowing of the leaves) and included all visible changes until 
after the spots had fallen. After sectioning, several stains were tried. 
Durand's (9) method for differentiating mycelium of parasitic fungi 
worked very well, and was used where it was desired to see the position 
ari*d extent of the mycelium. 

The mycelium is intercellular, with haustoria which penetrate the 
host cells (figs. 17, 18). The haustorium enters through a very small 
hole in the cell wall and is very much attenuated as it enters, but the 
end enlarges into an oval or elliptical body which contains a nucleus 
and a comparatively large vacuole. After the haustorium has entered, 
the protoplasm of the invaded cell often deposits a cellulose sheath 
around the haustorium, apparently similar to that formed around the 
haustoria of the Erysiphaceae as described by Smith (25). This sheath 
often extends along the wall of the host cell for some distance also. 

The host cells are not killed at first except those in contact with 
the stroma; and their death is probably brought about by drying 
rather than as a result of any toxic secretion from the fungus proto- 



150 BASCOMBE BRITT HIGGINS 

plasm. That no very poisonous toxin or enzyme is given off is indi- 
cated by the fact that cells penetrated by the haustoria appear healthy 
and are able to deposit a cellulose sheath around the haustorium. 

In every case examined acervuli had already formed and had 
broken the epidermis of the leaf, more commonly on the upper surface, 
but often on both surfaces. In every case also, even the very earliest, 
the tissue of the spot containing the fungus had already separated or 
begun to separate from the surrounding tissue. 

Formation of Absciss Layer Around the Spots. — This separation 
is brought about by the enlargement of a layer of cells at some distance 
from the ends of the mycelium (figs. 14, 15, 16). Their enlargement is 
so abrupt and so great that the active cells separate from the adjoining 
inactive cells inside. The enlarged cells have lost their chloroplastids 
and nuclei, and only a thin layer of protoplasm lines the wall. The 
loss of the chloroplastids causes the watery appearance of this ring of 
tissue which was noted by Duggar (8). The tendency for the forma- 
tion of this layer to follow the veinlets of the leaf as mentioned by this 
author has very rarely been noticed. The spots which drop out are 
usually very nearly round; and, even when quite irregular, the margin 
is evenly curved and smooth, not angular as it would be if the separa- 
tion was limited by the veinlets. 

After the separation is completed all around, the spot turns 
yellow, shrivels rapidly and soon drops out. Sometimes (often in P. 
serotina) the tension exerted by the enlarging cells is not sufficient to 
rupture the cuticle, but the cellular tissues are ruptured and the spot 
dries but remains in position. After the separation has occurred a 
layer of cells just outside the enlarged cells shrivel, forming additional 
protection for the healthy tissue. 

In order to obtain the earlier stages of the process, plants in the 
greenhouse were inoculated with fresh conidia from leaves of P. 
virgifiiana which had been inoculated with a pure culture from the 
same species. The conidia were placed in a drop of water on a glass 
slide and a little powdered chalk added for the purpose of marking the 
inoculated spots. Small droplets of this water containing spores and 
chalk were placed on the under surface of the leaves of two plants and 
on the upper surface of two, and all four plants covered with bell jars. 
After three days some of the inoculated spots were cut out, killed in 
hot (about 60° C.) chrom-acetic acid, and embedded in parafifin. 
This was repeated every 24 hours until acervuli appeared above the 



CYLINDROSPORIUM ON STONE FRUITS I51 

chalky spots on the sixth day. Infection did not occur in any case 
where the spores were placed on the upper surface of the leaves. 

When this paraffined material was cut and stained no mycelium 
was found within the leaf tissue killed the third day. By the end of 
the fourth day some germ tubes had entered and had formed quite 
extensive mycelium. When these mycelial threads, entering the 
stomates of the lower epidermis and traversing the mesophyll, come 
in contact with the upper epidermis they branch profusely and form 
by the end of the fifth day a very delicate stroma with conidiophores 
and young conidia. By the end of the sixth day conidia have matured 
in sufBcient numbers so that the pressure ruptures the leaf epidermis 
above the stroma. 

No haustoria were found until the fifth day. The host cells appear 
normal until the sixth day, when, in a few cells just beneath the stroma 
and in contact with it, the protoplasm was slightly shrunken, due 
probably to drying. This appearance spreads rather rapidly after 
the epidermis is broken. The leaf tissue containing the fungus is 
separated off by the enlargement of certain cells mentioned above and 
the separated tissue yellows rapidly. The length of time varies, but 
usually within 7-10 days after infection the spots begin to yellow and 
drop out. 

An apparently related case to this shedding of the diseased spots 
is that reported by Galloway (12) in which the needles of Piniis 
virginiana drop off after the formation of Coleosporium pustules. 
Galloway says that this casting is not caused by the fungus directly 
but by the loss of water through the break in the epidermis. 

Enlargement of Cells of Absciss Layer. — To explain the enlargement 
of the cells of the separation layer, four hypotheses have been sug- 
gested, viz.: First, the release of tension due to shrinking of the 
adjoining cells may allow the cells to enlarge; second, the cell walls may 
be softened by some enzyme secreted either by the fungus or by the 
host protoplasm, thus allowing the cells to expand; third, the colloids 
of the cell (protoplasm, cell wall, etc.) may be so modified as to have 
a greater affinity for water; ^.nd fourth, the osmotic pressure may in 
some way be increased in these cells. Of the four the last seems to 
be the most plausible, and while it is not yet proven beyond doubt 
all observations seem to support this as an explanation for the 
phenomenon. 

The first hypothesis is invalid because these cells begin to enlarge 



152 BASCOMBE BRITT HIGGINS 

before the adjoining cells have shrunken to any appreciable extent. 
Further the cells of these leaves do not enlarge to any appreciable 
extent, if the tension is removed entirely by cutting or tearing the 
leaf or if the tissue is macerated and' the cells set free in water. 

The second hypothesis does not appear valid, since if such an 
enzyme were secreted it would probably spread to adjacent cells. 
Also it is not very likely that, with the loss of water from the adjacent 
cells and consequently to some extent from these, the internal pressure 
would be great enough to expand the cells very much. At least with 
only ordinary osmotic pressure to hold the cell water, the cells would 
quickly collapse on exposure to the air. 

The third hypothesis is similar to that held by Fischer (lo) to be 
the cause of oedema in animals, where the swelling is said to be due 
to modifications in the fibrin of the blood, muscle, etc. At first the 
disappearance of the plastids and nuclei seemed to argue in favor of 
this hypothesis, but when the stained sections were examined again 
the interior of the cells was found to be colorless while the protoplasm 
lining the cell wall was stained and distinct, which indicates that the 
interior is filled with cell sap. 

That osmotic pressure is capable of causing the enlargement of 
plant cells is shown by the development of intumescences under various 
conditions during which increased osmotic pressure in the cells occurs. 
The question is then, how can the increased pressure be brought about 
in this case. 

Production of Shot Holes Correlated with Amygdalin Content of 
Leaves. — Observations seem to indicate that the shedding of the dis- 
eased spots is correlated with the amygdalin content of the leaves. 
As before mentioned the spots are shed from P. virginiana during all 
the spring and early summer. Morse and Howard (20) have shown 
that the young leaves of this species are very rich in amygdalin which 
diminishes in amount with the age and vigor of the leaves. Of the 
species under observation, the following also shed the spots infected 
by Cylindrosporium while the leaves are young: P. serotina, P. penn- 
sylvanica, P. cerasus and less frequently P. domestica. They are never 
shed from leaves of P. mahaleh, P. spinosa (not seen infected while 
leaves were very young) and, except under rare conditions, from P. 
avium. According to publications summarized by Wehmer (30), 
amygdalin is found in the leaves of P. virginiana, P. pennsylvanica, 
P. serotina, P. cerasus (?) and in young but not in mature leaves of 
P. domestica. It is not found in P. mahaleh, P. spinosa, or P. avium. 



CYLINDROSPORIUM ON STONE FRUITS I53 

It has been found that leaves which contain amygdaHn produce 
also an enzyme, emulsin, which, under certain conditions, breaks the 
amygdalin down into benzoic aldehyde, hydrocyanic acid and glucose. 

Morse and Howard (20) found that wilted cherry leaves yielded 
much more prussic acid than fresh leaves, but offer no explanation 
as to why the leaves should do so. 

More recently H. E. and E. F. Armstrong (2), in an interesting 
paper on "The origin of osmotic effects," find that prussic acid is set 
free in the leaves of the cherry laurel by treating the leaves with vapors 
of anaesthetics, with alcohols, or many other organic and inorganic 
compounds which enter the cells of the leaves. The authors think 
that when these substances enter the cells they change the osmotic 
relations in the cell, and enzymes are set free which break down the 
hydrolites stored there. 

Guignard (14), who has done much work on emulsin and cyano- 
genesis, found emulsin in the endodermis of the vascular bundles only 
in leaves of P. laiirocerasus. He suggests that wilting, action of 
chemicals, anaesthesis, or anything which alters the osmotic relations 
of the cells would bring the emulsin and hydrolite together. 

Mann (19)^ states that, in curing certain grades of tea, leaves 
allowed to wilt slowly increase the amount of the enzymes which break 
down the objectionable compounds. He thinks that these enzymes 
are not present as such, but zymogens are present which break down 
and form enzymes when the leaves wilt. 

Green (13) thinks that in many of the plants enzyme antecedents 
exist first as zymogens, and in a few instances has shown rather 
definitely that such is the case. 

It is quite possible that the emulsin zymogen exists, along with 
amygdalin in the cells of cherry leaves, and that the enzyme is set 
free by slight changes in the osmotic pressure in the cell. Should this 
be the case certain obser\'ed phenomena (e. g., the rapid splitting of 
amygdalin in wilted leaves) could be more readily understood. 

In whatever condition the enzyme exists, in cherry leaves it seems 
certain that it comes in contact with and breaks down the amygdalin 
when the leaf tissue wilts; and with this fact in mind a very plausible 
theory of "shot hole" formation can be formulated. When the 
acervuli and spores of Cylindrosporium break the leaf epidermis, the 

5 Original paper not seen but only a summary in Fowler's "Bacteriological 
and Enzyme Chemistry." 



154 BASCOMBE BRITT HIGGINS 

underlying cells dry out rapidly. As the wilting spreads the amygdalin 
comes in some way in contact with the emulsin and is broken down. 
Each molecule of amygdalin breaks down into four molecules (one 
each of hydrocyanic acid and benzaldehyde and two of glucose), 
thus materially increasing the osmotic pressure in the cells where this 
occurs. The increased osmotic pressure enables them to draw water 
from the adjoining cells and swell until this pressure is more nearly 
equalized. 

Since HCN has been found to increase the activity of proteolytic 
enzymes, it is quite probable that its presence in the enlarging cells 
causes the digestion of the plastids. Butkewitsch (7) found that 
addition of HCN accelerated the self digestion of the proteins in 
crushed seeds of several plants. Vines (29) also noted this property 
of HCN and suggested that its function in seeds might be to facilitate 
proteolysis of the reserve materials on germination. 

Amygdalin Removed from Area of Leaf Occupied by the Mycelium. 
— One question which naturally arises is, Why is this separation layer 
always formed at such a distance from the acervulus as to include the 
fungus mycelium? This question is readily answered according to the 
previously discussed theory, if the fungus is capable of using amygdalin 
as a food; since in that case the amygdalin from the cells in the im- 
mediate neighborhood of the mycelium would be absorbed by it. 
The separation layer would then be formed by the cells outside this 
region, the amygdalin of these cells breaking down and producing the 
necessary osmotic pressure. The fungus could certainly use a part 
of the amygdalin molecule (e. g., the sugar), if it contains an enzyme 
to split the amygdalin into these simpler compounds. 

To test this hypothesis, several cultures of the fungus from P. 
virginiana, P. mahaleb, P. pennsylvanica, and P. avium all growing on 
sterilized bean pods were used. From four cultures the liquid in 
the bottom of the tube was filtered off and a quantity of absolute 
alcohol added to the filtrate. A flocculent precipitate was soon 
formed. The liquid was again filtered, the precipitate washed with 
a mixture of ether and alcohol, dried and redissolved in 8 c.c. of water. 
The liquid from each tube was thus treated separately. As checks, 
the extract from two of the cultures was boiled and to each of the 
four, 2 cc. of a 5 per cent solution of amygdalin was added. A few 
drops of chloroform were added to each as an antiseptic. The prepara- 
tions were then corked and set in a locker in the laboratory. After 



CYLINDROSPORIUM ON STONE FRUITS 155 

1 8 hours there was an almond odor in the tubes containing the un- 
boiled extract and by the end of 8 hours this odor was very strong. 
At this time they were tested for reducing sugar and for HCN. Both 
were present in the tubes containing the unboiled extract while the 
checks gave no trace of HCN and only a slight trace of reducing sugar. 
The other cultures were crushed in a mortar, extracted with 30 per 
cent alcohol, filtered, and the filtrate treated in the same way that 
the liquid from the other tubes had been treated. In every case 
emulsin was found to be present in the unboiled extract. 

For comparison with the behavior of Cylindrosporium and its 
hosts, a Septoria found on leaves of P. pennsylvanica was studied. 
In this case the mycelium passes directly through the host cells and 
kills the host tissue before fruit bodies are formed. The spots drop 
out less frequently than Cylindrosporium spots on the same species. 
Where they do drop out however there is a separation layer formed in 
the living tissue just outside the dead tissue. A similar separation 
layer was also found in leaves of P. americana affected with Cercospora. 
(From herbarium material distributed as Cylindrosporium padi Karst. 
in Griffith's "West American Fungi" 75a.) 

Frank (11) also found that such a callus layer is formed around 
spots infected by Gnomonia erythrostoma Pers., and Duggar (8) 
noticed its formation in leaves of Prunus in which "shot hole" was 
produced by poisonous compounds. 

Development of the Perfect Stage^ 

The perfect or ascigerous stage of the fungus on P. avium was 
found in the spring of 191 1 and its development was studied more 
closely during the winter and spring of 1912. From the knowledge 
thus gained it has been comparatively easy to find and follow the 
development of the perfect stage on the other hosts under observation, 
especially since trees whose leaves were infected with Cylindrosporium 
were located during last summer (1912). It has been found on all the 
hosts under observation except P. spinosa, of which only a few leaves 
were found infected with the conidial stage last summer. 

There is considerable variation in the perfect stages found on the 

6 A brief description of the structure and development of the perfect stage of 

Cylindrosporium on P. avium was given in a previous note (16); but since many 

points of interest could not be included in a brief note, the entire development will 

be given in more detail here. 



156 BASCOMBE BRITT HIGGINS 

dififerent host species, but they may be divided into two main groups 
according to the shape of the ascocarp and its position in the leaf. 
Since the development varies slightly in the two types, the develop- 
ment on P. avium will be described and the others compared with this 
one. 

On this host the production of normal Cylindrosporium conidia 
(macroconidia) ceases usually early in August, and from this time 
until after the leaves fall minute conidia (microconidia) about one 
tenth the length of the macroconidia are formed in great numbers. 
They are abstricted from the apex of short branched conidiophores 
(fig. 39) which appear to be the same ones on which the macroconidia 
were produced earlier. At least they are on the same stroma and in 
similar positions. 

Almost simultaneous with this change in spore form, the stroma 
begins to develop downward through the mesophyll and palisade 
layers of the leaf. This growth is at first composed of separate but 
profusely branched and rather tightly packed threads; but an outer 
pseudoparenchymatous layer is later differentiated. The host cells 
are often surrounded by this mycelial growth and often, especially 
the lignified cells of the vascular bundles, remain so enclosed until the 
fruit body matures the next spring (figs. 6, 8). Before the formation 
of the pseudoparenchymatous covering, coils of densely staining 
hyphae appear in the stroma. Several of these coils, often six or 
eight, are formed in each stroma. Each coil consists of two or three 
turns in the hypha and is made up of several uninucleate cells. Its 
free end is extended as a trichogyne-like structure, which is also 
several-celled and slightly enlarged at the apex. This swollen tip 
extends above the surface of the stroma and ends just above the layer 
of conidiophores from the apex of which the sm*all conidia (spermatia ?) 
are being abstricted. Soon after leaf-fall the trichogyne-like struc- 
tures disintegrate and very soon disappear entirely. The fate of the 
coiled base of this structure is as yet an open question. The dense 
pseudoparenchymatous covering, which about this time develops 
entirely around the stroma, makes the inner portion extremely difficult 
to fix satisfactorily. In material killed November 7 in Flemming's 
weak osmic acid fixer, the coils show decided signs of degeneration. 
They are stained scarcely at all by Heidenhain's iron alum haematoxy- 
lin, when the surrounding cells are deep black. Also in some of the 
material killed later no sign of the coils can be seen. On the other hand, 



CYLINDROSPORIUM ON STONE FRUITS 157 

in some material from the same tree killed December 28 in Carnoy's 
alcohol acetic acid fixer, many stromata show apparently healthy coils. 
Later than this they were not seen with certainty. 

The pseudoparenchymatous covering of the stroma separates the 
layer of microconidiophores which now gelatinize and glue the rem- 
nants of the epidermal cells to the surface of the stroma. The cells 
of the pseudoparenchymatous covering as well as the mycelium in the 
leaf tissue become thick walled and dark colored. 

The stroma usually extends entirely through to the upper epi- 
dermis, but remains covered both above and below by the leaf epi- 
dermis. It is apparent only because of the dark color, and in this 
condition the fungus passes the winter. 

During the first warm days of March the stroma begins to swell 
toward the lower (dorsal) surface of the leaf, and when sectioned this 
swelling is seen to be due to a row of erect parallel hyphae which later 
are seen to be paraphyses. The asci do not appear until about the 
first of April or later. They develop from branched ascogenous hyphae 
which arise near the base of the stroma. During the latter part of 
April and the first of May the asci enlarge rapidly and lift the covering 
until it finally breaks in a more or less stellate manner. The break 
occurs before the ascospores are mature, but they mature in a very 
short time thereafter. 

The asci open by a pore in the papillate apex and the spores are 
shot out. On taking leaves, in which the ascospores are just mature, 
from a moist chamber, clouds of spores have a few times been seen 
shot out from the under surface. 

After the ascospores are shed the asci and paraphyses disappear, 
and long slender conidia are formed on short conidiophores which 
arise apparently as branches from the base of the paraphyses. They 
are once or twice septate and resemble Cylindrosporium conidia but 
are usually longer and a little more slender. 

Besides being on P. avium this type of fruit body and development 
was found on P. pennsylvanica and P. cerasus. 

In the other type, found on P. domestica, P. insititia, P. virginiana, 
P. serotina and P. mahaleh, the stroma develops beneath the lower 
epidermis and does not extend into the leaf tissue to any appreciable 
extent. The development is more outward, thus protruding and form- 
ing rather prominent disk-shaped to flattened-globose bodies on the 
under side of the leaf. 



158 BASCOMBE BRITT HIGGINS 

In the leaves of P. serotina, P. virginiana and P. mahaleb the stroma 
never becomes black but changes to a yellowish brown or to a dull 
orange color with a waxy appearance when wet. The paraphyses 
are differentiated much earlier (in January or February) and the 
ascospores mature slightly earlier. Leaves of P. serotma received 
from Clemson College, S. C, on March 13, bore mature fruit bodies. 
In leaves from the same tree received in the fall and wintered over in 
wire cages (Ithaca), they did not mature until about a month later. 

On the plum (P. insititia and P. domestica) the stromata often 
turn coal black before the leaves fall. They protrude from the 
under surface of the leaf and because of the black color are very 
prominent all during the winter. The spring development is much 
slower and later than in P. virginiana, and slightly later than in P. 
avium. In all other respects the general development as far as observed 
was similar to that described on P. avium. 

Infected leaves of all the host species were brought into the labora- 
tory at intervals during the fall and winter. For some reason, how- 
ever, the fruit bodies refused to develop if brought in before the para- 
physes had been differentiated. Perhaps the excessive moisture, or 
else lack of freezing, prevented the formation of the ascogenous hyphae. 
It was also noticed that the fruit bodies failed to develop outside when 
the leaves were packed closely together and therefore moist and poorly 
aerated, and also when a leaf was folded so that half of its under surface 
was next the ground the fruit bodies failed to develop on the side in 
contact with the ground. 

When plum leaves were kept very moist in a closed moist chamber 
the asci did not develop, but conidia were formed instead. This 
may have been due to the inherent nature of the fruit body, but that 
it was due to the effect of excessive moisture is more probable. Leaves 
which had been kept very wet until most of the fruit bodies had 
developed into conidia-bearing structures, were gradually dried by 
exposing to the air for a few minutes each day when normal asci and 
paraphyses developed. 

Relation of the Ascigerous Stage to Cylindrosporium 

In order to remove all doubt as to the genetic connection between 
this ascogenous fungus and the Cylindrosporium which is parasitic 
on the several host species, several series of inoculations and cross 



CYLINDROSPORIUM ON STONE FRUITS 1 59 

inoculations were made during the early spring of 1912 and 1913. 
The inoculations were all made in the greenhouse and mostly before 
the ascospores had developed outside. 

Species of Primus Employed in the Inoculatio?!s. — The plants used 
for inoculating in 1912 were imported seedlings of mazzard (P. avitim 
furnished by the J. B. Stewart Nursery Co.) and small trees of P. 
serotina obtained from a nearby thicket. These were cut back to mere 
stubs, dipped for a few minutes into a 7 per cent CuSOi solution, 
planted in small pots, and set in the greenhouse. 

In 1 913 these plants were given the same treatment and used 
again. About 50 trees each of P. virginiana and P. pennsyhanica 
and 15 of P. americana obtained growing wild in this region; 50 
mahaleb cherry and 50 myrobalan plum trees furnished by the Green- 
ing Nursery Co.; 50 sour cherry (Early Richmond) and 50 peach 
(Elberta) trees bought from a local nursery; and 100 plum trees, 25 
each of P. domestica (several varieties), P. americana (several varieties), 
P. hortulana (several varieties), and P. insititia, furnished by the 
Horticultural Department of the Geneva Experiment Station were 
given the same treatment. 

The trees were cut back so as to give vigorous healthy leaves for 
inoculating and also so they could be covered with bell-jars when this 
was desirable. They were dipped in the CuSO^ solution to kill any 
conidia of Cylindrosporium as well as spores of other fungi which 
might interfere with the results of inoculations, although in so far as 
Cylindrosporium is co icerned sterilization was probably unnecessary, 
since all observations indicate that the conidia are very short lived 
and are unlikely to live over winter. None of the plants were ever 
attacked by Cylindrosporium unless inoculated, although often kept 
as checks under conditions very favorable for infection had conidia 
been present. 

The plants when used for inoculation had vigorous shoots with 
usually 12-50 leaves. The myrobalan plums however were so slow 
in starting that only a few small plants were ready when the last 
inoculations were made. 

Inoculations with Conidia. — The first series of inoculations was 
made for the purpose of determining the conditions most favorable 
for infection. Four plants of mazzard cherry were inoculated with 
a pure culture of Cylindrosporium from the same host species. The 
conidia were shaken up in a small amount of sterile water which was 



l60 BASCOMBE BRITT HIGGINS 

then placed in small droplets on the upper surface of the leaves of 
one plant, and on the under surface of the leaves of the other three. 
The former and one of the latter were then covered with bell-jars. 
Two of those with conidia on the under surface of their leaves were 
left in the open air of the greenhouse, but moist absorbent cotton 
was wrapped around the leaves of one plant. 

At the end of ten days the plants were examined, when several 
Cylindrosporium acervuli were found on the two plants under bell- 
jars, a few on the plant whose leaves were wrapped with moist cotton, 
but none on the plant whose leaves were left exposed to the air. 

This showed clearly that the leaves must be kept moist for satis- 
factory infection to occur; so the bell-jar method was used in all later 
inoculations except where otherwise stated. 

In the next series ascospores were used. Ascocarps which had 
matured on leaves placed in a moist chamber March i6 were on April 
4 crushed in a small amount of sterile water which, after being ex- 
amined microscopically and found to contain quantities of ascospores, 
was used for inoculating several plants. When examined 7 days later 
numerous small white specks, which proved to be acervuli of Cylindro- 
sporium, were found on the under side of the leaves. 

On April 16 two more plants were inoculated in the same way 
and at the end of eleven days 45 acervuli were found on their leaves. 

To determine whether conidia produced in the ascocarps also 
served to propagate the fungus, these were used to inoculate some 
plants. The conidia, if leaves are kept moist after the ascospores are 
shed, collect in small white clusters on top of the old ascocarps. 
Several of these clusters were picked up with a needle and distributed 
in drops of water on sterile glass slides. The water was then examined 
under the microscope and found to be apparently free from ascospores 
which can be distinguished by their smaller size and blunt ends. 
The two plants which were then inoculated with these conidia showed 
several acervuli at the end of seven days. 

Because of the difficulty of obtaining any mycelial growth, no pure 
cultures were ever obtained from these apothecial conidia from P. 
avium. They frequently germinated in water and in agar but when 
the germ tubes had reached less than half the length of the spore, 
growth ceased. 

Inoculations ivith Ascospores. — The ascospores behaved in much 
the same way. Several hundred cultures were made during the spring 



CYLINDROSPORIUM ON STONE FRUITS l6l 

of 191 1 and 1912 before any growth was obtained. Spores were often 
found germinating but when transferred to fresh agar, steriUzed bean 
pods, or sterile sUces of pear, no further growth occurred. Bean, 
potato, malt extract, prune, cherry leaf, and plain agar (agar dissolved 
in tap water), were tried. The plain agar was finally adopted for 
all germination trials since other fungi and bacteria developed to a 
much less extent and the ascospores (which germinated more freely 
than in the other agars) could more often be obtained free from 
contamination. Finally from a single lot of cultures 37 colonies 
developed from germinating ascospores transferred, some to tubes of 
plain agar, others to steamed bean pods and slices of pear. 

The growth was very slow. Nearly a month elapsed before any 
growth could be seen with the unaided eye. The colonies developed 
as small oval masses similar to those of Cylindrosporium. At first 
great quantities of conidia were produced, but after about two months 
conidial production ceased and the stroma turned dark in most of the 
colonies. Transfers from this stromatic material gave few conidia so 
that difficulty was experienced in obtaining infections the next winter. 

The first inoculations with these pure cultures were made Feb. i, 
1913. A colony from a steamed bean pod was crushed in some sterile 
water and used to inoculate two plants. After two weeks no infection 
was found and the same plants were again inoculated this time with 
spores produced on a very young colony. The drops of liquid left 
discolored spots where placed on the leaves and after seven days 40 
of these spots showed Cylindrosporium acervuli. 

Ascocarps were not found on the other species of Prunus until the 
spring of 1913; so, although several attempts have been made, no 
pure cultures have been obtained from them. Successful inoculations 
have been made with ascospores from leaves of P. virginiana, P. sero- 
tina, P. pennsylvanica, P. domestica, and P. insititia and with a pure 
culture from apothecial conidia from P. insititia infection has been 
produced on P. americana. 

In fact the ascospores are more active in producing infection than 
the conidia of Cylindrosporium. Ascospores from leaves of P. 
virginiana produced abundant infection on plants of P. serotina and 
P. virginiana while conidia formed on the P. virginiana plants from this 
inoculation would not cause the disease on P. serotina although several 
plants were inoculated under exactly the same conditions, and abun- 
dant infection occurred on a plant of P. virginiana inoculated at the 



1 62 BASCOMBE BRITT HIGGINS 

same time. In like manner the ascospores, but not the CyHndro- 
sporium conidia from P. serotina, would infect P. virginianum or P. 
mahaleh plants. 

Plants of P. serotina, P. virginiana, and P. americana have several 
times been inoculated with ascospores and conidia from P. avium 
but no infections have ever been obtained from such inoculations. 
Likewise no infection has ever been produced on P. avium by either 
ascospores or conidia from P. virginiana or P. seroti^ia. 

During the last weeks of April of this year a large series of cross 
inoculations were made in another greenhouse on all the different 
species before mentioned; but these resulted in almost total failure, 
probably because of the low temperature of the house. No infections 
were obtained except with the organism from P. virginiana and P. 
serotina. Only the Cylindrosporium conidia from P. virginiana were 
used and no infections were obtained except on P. virginiana where 
infection was rather abundant. Both ascospores aa^ conidia of P. 
serotina were used. From the conidial inoculations infections occurred 
on P. serotina only. The ascospores however produced infection on 
P. serotina, P. virginiana and P. mahaleb. 

The leaves pi P. virginiana have also been infected with a pure 
culture obtained from conidia of the Cylindrosporium on the fruits 
of the same species. This shows that one species is capable of infecting 
both leaves and fruit of this host. 

Systematic 

The study of morphological and cultural characters of Cylindro- 
sporium from the various host species showed some slight variations, 
but none which were prominent or constant enough to be of specific 
value. It was thought, therefore, before a comparison of the perfect 
stage from the different hosts was made, that there was but a single 
species with several more or less distinct forms on these hosts. 

Relation of Species of Cylindrosporium to the Natural Subdivisions 
of Primus. — A careful study of the ascogenous stage from the different 
hosts showed that there were marked and constant differences by 
which the forms might be divided into three distinct groups. The 
first group includes all those which have the type of development 
found on P. avium (in which the ascocarp extends from one epidermis 
of the leaf to the other in contrast with those in which the ascocarp is 



CYLINDROSPORIUM ON STONE FRUITS 163 

only subepidermal). They are distinct in the shape of the ascocarp, 
its position in the leaf tissue, and in the shap^and size of the asci. 

Those having the subepidermal ascocari^ fall into two distinct 
groups. On the plums (P. domestica and P. insititia) the fruit bodies 
are coal black with a very decided tendency to be aggregated in clus- 
ters, the asci are smaller, more slender than in the other forms, and are 
almost filled by the spores which are more slender than in either of 
the other forms. 

Contrasted with this we find on P. virginiana, P. serotina, and P. 
mahaleh the light (yellowish brown to dull orange) colored fruit 
bodies, larger asci, and apothecial conidia much larger than in the other 
forms. The covering of the fruit body is composed of more delicate, 
thinner-walled cells. 

After this grouping was made it was found that the hosts of each 
group fall in the same natural group of the genus Prunus, with the 
possible exception of P. mahaleb. The hosts of the first group are all 
in the subgenus Cerasus of Engler and Prantl, with flowers in umbels. 
Those of the second group are plums which are placed in the subgenus 
Prunophora by the same authors, while those of the third, with the 
exception of P. mahaleb, are placed by them in the subgenus Padus 
which has the flowers in elongated racemes. P. mahaleb has its 
flowers in a short raceme, and is placed by them in the subgenus 
Cerasus but in a group separate from the other members of the sub- 
genus. Britton places it in a group with P. virginiana and P. serotina 
all having flower clusters terminating branches of the present year's 
growth. 

The results of cross inoculations show a correlation of hosts with 
characters of the ascogenous stage. In no case has it been found 
possible to transfer, by inoculations, the fungus from hosts of one'group 
to hosts of another group. 

The fact that the hymenium of the ascocarp is surrounded — until 
nearly mature — by a wall of tissue which finally opens in a stellate 
manner places all forms of the fungus in the Phacidiales. The relation 
of the ascocarps to the leaf tissue (covered by the adherent host epi- 
dermis), the shape of the asci, and the elongated colorless spores 
grouped in a fascicle show a distinct relation to Coccomyces in which 
genus therefore all forms are included. Had only the light-colored 
form on the Padus group been found there might have been some 
question as to its relation to this genus, since Coccomyces is said to 



164 BASCOMBE BRITT HIGGINS 

have black ascocarps; but the fact that three forms, so evidently 
related as those under consideration, show such wide variation in 
color indicates that thexolor of the ascocarp is here not a character of 
generic rank. Because, however, of the morphological and biological 
differences, before mentioned, the forms are divided into three species, 
as follows: Coccomyces hiemalis to include the forms on Prunus avium, 
P. cerasus, and P. pennsylvanica; Coccomyces prunopkorae n. sp. to 
include the forms on the plums (P. americana, P. domestica and P. 
insititia) ; and Coccomyces lutescens n. sp. to include the forms on P. 
serotina, P. virginiana, and P. mahaleb. For these species the following 
characterization is given: 

DESCRIPTION OF SPECIES 

Coccomyces hiemalis Higg'ins, Science, N. S. 37: 637 and 638. 1913. Ascocarps 
embedded in the tissue of the leaf — usually filling the entire space between the lower 
and the upper epidermis — both of which usually adhere to the wall of the ascocarp, 
scattered to^ubaggregate, ovate to orbicular, dark brown or black, at first closed 
but at maturity opening by irregular stellate slits on the under side of the leaves, 
125-2 lO/u in diameter; hymenium pale gray to flesh-colored; asci clavate with a long 
stout pedicellate base, and abruptly papillate apex, 8-spored, 70-95 X 11-14^1; 
paraphyses filiform, septate, apex slightly enlarged, often hooked, often forked; 
ascospores linear, 33-50 X 3.5-4-5M (the smaller size given in original description 
was due to a typographical error), continuous or 1-2 septate, fascicled in large end 
of the ascus; apothecial conidia produced on short conidiophores in apothecia 
after shedding of ascospores, long, slender, 50-80 X 2.5-4/u, curved, continuous or 
1-2 septate. 

Conidial stage {Cylindrosporium hiemalis): Mycelium intercellular with small 
haustoria which penetrate the host cells; spots small, brown or reddish brown, some- 
times dropping out and producing "shot holes"; acervuli amphigenous or more 
commonly hypophyllous, subepidermal, finally erumpent exposing the spores; 
conidia elongate, curved or flexuous, 45-65 X 2.5-4^1, continuous or 1-2 septate; 
microconidia (spermatia?) produced in same acervulus in late summer and fall, 
small, continuous 4-5 X i.5m- 

Conidial stage parasitic in leaves of P. avium, P. cerasus, and P. pennsylvanica. 
Ascigerous stage saprophytic, appearing the last of April to June, on fallen leaves of 
the same hosts following the conidial stage. 

Latin diagnosis: Ascomatibus hypophyllis sparsis interdum subaggregatis, 
innatis, punctiformibus, fuscis vel nigris, ovatis vel orbicularibus, 125-250// lat., 
primum clausis, deinde in lacinias plures acutas dehiscentibus; disco pallido carneo 
vel grisea; ascis clavatis crassiuscule stipitatis, 70-90 X I1-14M, octosporis, apice 
papillato; paraphysibus filiformibus, simplicibus aut ramosis, apice recto aut curvato; 
sporidiis fasciculatis, linearibus 35-50 X 3.5-4.5M, simplicibus aut 1-3 septatis; 
conidiis in apotheciis filiformibus, flexuosis, 50-80 X 2.5-4^, 1-2 septatis. 

Hab. In dejectis foliis Prunii avii, P. cerasi, et P. pennsylvanicae. 



CYLINDROSPORIUM ON STONE FRUITS 165 

Status conidicus: maculis sparsis vel confluentibus, minutis, brunneis aut 
rufis-brunneis, interdum majusculis dejectis, acervulis solitariis amphigcnis, sub- 
epidermicis, disciformibus; conidiis filiformibus, flexuosis, denique emergcnti-supcr- 
ficialibus, hyalinis 45-65 X 2.5-41X, simplicibus aut 1-2 septatis; conidiis minoribus 
autumno, hyalinis, continuis 4-5 X i.5m- 

Hab. In foliis vivis Prunii, P. cerasi, et P. pennsylvanicae. 

Coccomyces pnmophorae n. sp. Ascocarps hypophyllous, usually aggregated, 
subepidermal, erumpent, disk-shaped to subglobose, 125-250 X 100-160^, black, 
at first closed but at maturity opening in a stellate manner; hymenium light gray; 
asci clavate to clyindrical-clavate, 63-87 X 9-12^1, opening by a pore at the papillate 
tip; spores slender, straight, curved near the end, 40-60 X 2.5-3.5JU, at maturity 
almost filling the asci, continuous or 1-3 septate; paraphyses simple or branched, 
enlarged at apex, septate, about as long as asci; apothecial conidia produced on 
short conidiophores inside the apothecia, following the asci, usually once septate, 
40-60 X 2.5-3.5JU, resembling ascospores but usually stouter. 

Conidial stage {Cylindrosporium pnmophorae) : mycelium intercellular with 
haustoria which enter host cells; spots small, brown or reddish brown, sometimes 
dropping out, producing "shot holes." Acervuli subepidermal, amphigenous, 
finally breaking through exposing the white mass of spores, conidia elongate, slender, 
straight or curved, 46-65 X 3-5-5/". usually i-septate; microconidia (spermatia?) 
found in same acervuli in late summer, small, continuous 4-5 X i.5m- 

Conidial stage parasitic in leaves of plums (P. domestica, P. insititia, P. americana'' 
and probably P. spinosa). Ascigerous stage saprophytic on fallen leaves of the same 
hosts, May to June, following the conidial stage of the previous summer. 

Latin diagttosis: Ascomatibus hypophyllis, aggregatis vel sparsis, subepidermicis, 
erumpentibus, disciformibus vel subglobosis, 125-250 X 100-160^, nigris, primum 
clausis deinde in lacinias plures acutas dehiscentibus; disco pallido griseo; ascis 
clavatis vel cylindraceoclavatis, fere sporidiis completis, 63-87 X 9-12^1, octosporis, 
apice papillato; paraphysibus filiformibus, simplicibus aut ramosis, septatis; sporidiis 
fasciculatis, linearibus 40-60 X 2.5-3.5JU aut 1-3 septatis; paraphysibus simplicibus 
aut continuis ramosis; sporidiis in apotheciis linearibus rectis vel flexuosis, 40-60 X 
2-5-3-5M, I septatis. 

Hab. in dejectis foliis Prunii domesticae, et P. instititiae. 

Status conidicus; maculis sparsis, vel confluentibus, brunneis aut rufis-brunneis 
interdum majusculis dejectis; acervulis solitariis, amphigenis, subepidermicis, 
disciformibus, conidiis filiformibus, rectis vel flexuosis, denique emergenti-super- 
ficialibus, hyalinis, 46-65 X 3.5-5^1 i septatis, conidiis minoribus autumno hyalinis 
continuis, 4-5 X 1.5^. 

Hab. In foliis vivis Prunii domesticae, P. insititiae, P. spinosae, et P. amer- 
icanae. 

Coccomyces lutescens n. sp. Ascocarps hypophyllous, subepidermal, erumpent, 
disk-shaped to flattened elliptical, 130-300 X 70-150/x, often depressed above before 
maturity, lutescent or dull orange in color, at first closed, then opening in an ir- 

^ Since infection and the Cylindrosporium acervulus can be produced on P. 
americana by the fungus from P. insititia, it is very likely that the perfect stage 
also develops on this species. 



l66 BASCOMBE BRITT HIGGINS 

regularly stellate manner, hymenium grayish to creamy white; asci clavate 70-80 X 
14-19/X, opening by a pore in the papillate apex; spores elongated, fascicled in end of 
ascus, 40-51 X 3.5-4-5^, continuous or occasionally once or twice septate; para- 
physes usually simple though occasionally branched, slightly enlarged at tip, about 
as long as asci; apothecial conidia very long, 58-87 X 3-5-5M, once-septate with 
usually a single vacuole in each cell. 

Conidial stage {Cylindrosporium lutescens) : spots small, brown to reddish brown, 
in early summer dropping out from leaves of some species and producing rounded 
"shot holes"; mycelium intercellular, with haustoria which penetrate the host cells; 
acervuli subepidermal, amphigenous; conidia exuding in masses or in long tendrils 
from the break in the host epidermis, long, slender 50-87 X 3-5-5/". continuous or 
1-3 septate, microconidia (spermatia?) produced in the same acervuli in late summer, 
Small, continuous, 4-5 X im- 

Conidial stage parasitic in leaves of P. serotina and in leaves and fruits of P. 
virginiana, and P. mahaleb. Ascigerous stage saprophytic on fallen leaves of the 
same species in May and June following the conidial stage of the previous summer. 

Latin diagnosis: Ascomatibus hypophyllis, sparsis, subepidermicis, erum- 
pentibus, disciformibus, 130-300 X 70-150^, luteis vel ferrugineis, primum clausis 
deinde in lacinias plures acutas dehiscentibus; disco pallido carnco vel griseo; ascis 
clavatis, crassiuscule stipitatis, 70-80 X 14-19^, octosporis, apice papillato; para- 
physibus, filiformibus, simplicibus aut ramosis; sporidiis fasciculatis, linearibus, 
35-50 X 3.5-4.5^, simplicibus aut 1-3 septatis; conidiis in apotheciis filiformibus, 
flexuosis, 50-80 X 2.5-4/i, i-septatis. 

Hab. in dejectis foliis Pruni serotinae, P. virginianae et P. mahalebis. 

Status conidicus; maculis sparsis vel confluentibus minutis brunneis aut rufis- 
brunneis, interdum majusculis dejectis; acervulis solitariis, amphigenis, subepider- 
micis, disciformibus; conidiis, filiformibus, flexuosis, denique emergenti-super- 
ficialibus, hyalinis, continuis 50-87 X 3-4-5^, i septatis; conidiis minoribus autumno 
hyalinis, continuis 4-5 X 1.5. 

Hab. in foliis vivis Prunii serotinae, P. virginianae, et P. mahalebis. 

General Discussion 

There are many points of interest in the hfe history and develop- 
ment of the Cylindrosporium on stone fruits, not the least of which is 
their relation to the host tissue. The parasitic stages of Ascomycetes, 
other than the Perisporiales which have epiphytic mycelium, usually 
kill the host tissue outright. In two notable exceptions to this, 
Gfionwnia erythrostoma Pers., and Polystigma riihrnm (Pers.) D. C. 
no haustoria have been found. Haustoria have been described for a 
large number of the Perisporiales (in the Erysiphaceae by many 
workers and by Maire (18) for Meliola^ and Asterina). In one of the 
Erysiphaceae, Oidiopsis taurica Salmon, the mycelium is endophytic, 
and it is partly so in Phyllactinia corylea (see Palla (21) Smith (25) 
8 Meliola is placed in the Perisporiales by Saccardo. 



CYLINDROSPORIUM ON STONE FRUITS 167 

Salmon (24)); but even here the host tissue is not killed. In this 
respect Cylindrosporium resembles the last named species, and the 
presence of haustoria here is probably related to this behavior. 

The formation of spermatia-like bodies in connection with struc- 
tures which appear to be homologous with the ascogonial branches of 
the Collemaceae is also very interesting. The function of these bodies 
IS not known. Several attempts to germinate them have always re- 
sulted in failure, but considering the difficulty of germinating the 
normal conidia this is not a very strong argument in favor of their 
spermatial nature. Their position on the surface of the stroma with 
the trichogyne of the ascogonium (?) projecting among them would 
certainly facilitate fertilization, if they do now function or have ever 
functioned as sexual organs. The most nearly similar arrangement 
is found in species of Physma. Here there are several ascogonial 
branches arising in the base of the spermogonium, as in Coccomyces on 
stone fruits, but passing around the spermogonium to the surface of 
the thallus. See Stahl (27), Sturgis (28), and others. 

The ascogonial structures found in Gnomonia erythrostoma have 
been studied by Brooks (6) and were not found to function as female 
sexual organs. On the contrary he thinks the ascogenous hyphae 
arise from vegetative cells. A similar condition was found in Poly- 
stigma rubriim by Blackman and Wellsford (5). From analogy and 
from the fact that the ascogonial structures appear to disintegrate 
in many cases, one is led to think that in Coccomyces also the asco- 
gonium does not function as a sexual organ. However the fact that 
the ascogenous hyphae arise from several points similarly situated in 
the ascocarp indicates that at least a part of. the structure may function 
as an ascogonium. 

Control Measures 

Now that the complete life history of the fungus is known, it should 
be much easier to devise methods for controlling the disease. Since 
it lives over winter in the dead leaves it is very important that leaves 
from trees which are infested with the disease be raked together and 
burned or buried. If all such diseased leaves are destroyed in and near 
the cherry orchard there is little danger of the disease appearing the 
following season in such abundance as to be of a serious nature. 

If for any reason destruction of the diseased leaves is not feasible 
or desirable and one must depend on sprays to keep the disease in 
check, the spraying should begin early, at least by the middle of May. 



l68 BASCOMBE BRITT HIGGINS 

The only common wild species in this region which may harbor 
the fungus which attacks the sweet and sour cherries is P. pennsyl- 
vanica. 

There appears to be no danger of the disease passing from the wild 
choke cherry or the wild black cherry (P. serotina) to the sweet or sour 
cherry; but the fungus from the former may infect nursery seedlings 
of P. mahaleh. 

The fungus on the plums has so far been found to infect nothing 
but plums, but some of the wild plums, certainly P. americana, may 
harbor the fungus. 

Conclusions 

1. There are at least three species of Cylindrosporium parasitic 
on species of the genus PrunuS, which in their conidial stage resemble 
each other very closely. Whether or not Cylindrosporium padi Karst. 
also occurs in North America is not known; but from the fact that it 
occurs on P. padus, which has racemose flower clusters similar to P. 
serotina and P. virginiana, it is perhaps the same as that occurring on 
the two last named species. 

2. The conidial stroma of Cylindrosporium on Prunus develops 
centrifugally and is never turned up at the edges so as to resemble a 
pycnidial structure. 

3. The mycelium of the species of Cylindrosporium studied is 
intercellular and obtains its food, in part at least, by means of haus- 
toria which penetrate the host cells. Very often a cellulose sheath is 
then deposited around the haustorium by the host protoplasm. It 
seems that no toxin or substance injurious to the host protoplasm is 
secreted by the fungus, but a few of the host cells are killed probably 
by drying. 

4. "Shot hole" formation in the leaves of the host is apparently 
correlated with the presence of amygdalin. The amygdalin molecule 
breaks down into simpler molecules thereby probably increasing the 
osmotic pressure which causes the cells surrounding the spot to enlarge 
forming the separation layer. 

5. Beside the Cylindrosporium conidia three other spore forms 
are found in the life cycle of the species studied, viz.: microconidia 
(spermatia-like bodies), ascospores, and apothecial conidia. All of 
these except the microconidia are known to propagate the fungus on 
living leaves. 



CYLINDROSPORIUM ON STONE FRUITS 1 69 

6. The microconidia are not produced in pycnidial structures, as 
reported by Arthur (4) and by Pammel (22), but on the surface of 
the conidial stroma. 

7. While the microconidia (spermatia?) are being formed on the 
surface of the stroma, ascogonia-like structures are formed with their 
free end (trichogyne?) projecting above the surface. If these struc- 
tures do now function or have ever functioned as sexual organs, 
their formation at the same time and on the same stroma would make 
fertilization more certain. 

8. The fungus passes the winter as a stroma-like body in the fallen 
leaves, which in the early spring develops into an apothecium of the 
Phacidiaceous type. 

9. That the ascocarps are genetically connected with Cylindro- 
sporium has been shown by their continuous development from the 
stromata of Cylindrosporium, and by producing infection and Cylin- 
drosporium acervuli in living leaves when inoculated with ascospores 
from leaves, or with conidia from pure cultures from these ascospores. 

10. The forms of Coccom^ces found on the eight species of Prunus 
fall naturally into three species, one on each of three more or less 
natural subdivisions of the host genus. 

The study on which this article is based has been carried on in the 
Botanical Laboratory of Cornell University under the direction of 
Prof. Geo. F. Atkinson, whom I wish to thank for many helpful sugges- 
tions and criticisms. Acknowledgments are also due Prof. W. D. 
Bancroft for some valuable suggestions, Prof. J. G. Hall for material 
collected at Clemson College, S. C, the Geneva Agricultural Station, 
the J. B. Stewart Nursery Co., and the Greening Nursery Co., for 
plum and cherry trees contributed for experimental purposes. 

BIBLIOGRAPHY OF PAPERS CITED 

1. Aderholdt, R. Uber die Spriih- und Diirrfleckenkrankheiten (syn. Schusslocker- 

krankheiten) des Steinobstes. Landw. Jahrb. 30: 771-830, pi. 18. 1901. 

2. Armstrong, H. E. and E. F. The origin of osmotic effects. III. The function 

of hormones in stimulating enzymatic change in relation to narcosis and the 
phenomenon of degeneration and regenerative change in living structures. 
Proc. Roy. Soc. (London) ser. B. 82: 588-602. 1910. 

3. The function of hormones in regulating metabolism. Ann. Bot. 25^: 

507-519. 1911. 

4. Arthur, J. C. Plum leaf fungus. N. Y. Agr. Exp. Sta. Kept. 5: 293-298, 

figs. 6-10, 1886. Idem 6: 347, 348. 1887. 



I70 BASCOMBE BRITT HIGGINS 

5. Blackman, V. H. and Wellsford, E. J. The development of the perithecium of 

Polystigma rubrum D. C. Ann. Bot. 26: 761-767, pi. 70, 71. 1912. 

6. Brooks, F. T. The development of Gnomonia erythrostoma Pers. Ann. Bot. 24: 

585-605, pi. 48, 49. 1910. 

7. Butkewitsch, Wl. Uber das Vorkommen eines proteolitischen Enzyms in ge- 

keimten Samen und iiber seine Wirkung. Zeitschr. f. Physiol. Chem. 32: 

1-53- 1901. 

8. Duggar, B. M. The shot hole effect on the foliage of the genus Prunus. Proc. 

Soc. Prom. Agr. Sci. 19: 64-69. 1898. 

9. Durand, E. J. The differential staining of intercellular mycelium. Phyto- 

pathology 1: 129, 130. 191 1. 

10. Fischer, M. H. Oedema. Pp. 209, 1909 (Reprinted from the "Transactions of 

the College of Physicians of Philadelphia"). 

11. Frank, B. Die jetzt-herrschende Krankheit der Siisskirschen im Altenlande. 

Landw. Jahrb. 16: 401-436, pi. 2, 1887. 

12. Galloway, B. T. A rust and leaf casting of pine leaves. Bot. Gaz. 22: 433-453, 

pi. 22, 23. 1896. 

13. Green, J. R. The soluble ferments and fermentation. Sec. ed. 1901. 

14. Guignard, L. Sur la localisation dans des Laurier Cerise des principes qui 

furnissent I'acide tyanhydrique. Jour, de Bot. 4: 21-27, figs- i. 2. 1890. 

15. Influence de I'anesthesie et du gel sur le dedoublement de certains 

glucosides chez les plants. Compt. rend. Acad. "Sci. Paris. 149: 140-142. 
1909. 

16. Higgins, B. B. The perfect stage of Cylindrosporium on Prunus avium. Science 

N. S. 37: 637, 638. 1913. 

17. Karsten, P. A. Cylindrosporium padi Karst. (n. sp.). Mycol. Fenn. 16: 159. 

1884. 

18. Maire, R. Les sucoirs des Meliola et des Asterina. Ann. Myc. 6: 124-128, 

figs. 1-4. 1908. 

19. Mann, H. H. The fermentation of tea. Indian Tea Association, pamphlet 

pp. 22. 1906. 

20. Morse, F, W. and Howard, C. D. Poisonous properties of wild cherry leaves. 

N. H. Agr. Exp. Sta. Bui. 56: 113-123. 1898. 

21. Palla, E. iJber die Gattung Phyllactinia. Ber. d. Deutsch. Bot. Ges. 17: 64- 

72, pi. 5. 1899. 

22. Pammel, L. H. Spot disease of cherries. Iowa Agr. Exp. Sta. Bui. 13: 55-66. 

1891. 

23. Peck, Chas. H. Cylindrosporium padi Karst. Rep. State Botanist, State of 

N. Y. 48: 15. 1895. 

24. Salmon, E. S. Preliminary note on an endophytic species 01 the Erysiphaceae. 

Ann. Mycol. 3: 82, 83. 1905. 

25. Smith, Grant. The haustoria of the Erysipheae. Bot. Gaz. 29: 153-184, pis, 

II, 12. 1900. 

26. Sorauer, P. Pflanzenkrankheiten, third ed. 2: 1908. 

27. Stahl, E. Beitrage zur Entwickelungsgeschichte der Flechten, pt. I, pp. 55, 

pis. 1-4. 1877. 



CYLINDROSPORIUM ON STONE FRUITS I7I 

28. Sturgis, W. C. On the carpological structure and development of the CoUe- 

maccae and allied groups. Proc. Amer. Acad. Arts and Sci. 25: 15-52, pis. 
1-8. 1890. 

29. Vines, S. H. Tryptophane in proteolysis. Ann. liot. 16: 1-22. 1902. 

30. Wehmer, C. Pflanzcnstoff. (Phanerogamcn). 191 1. 

DESCRIPTION OF PLATES XIII-XIV 
Plate XIII 

Fig. I. Photograph of leaf of PrunUs avium with ascocarps, magnified about 
2 diameters. ^ 

Fig. 2. Photograph of a portion of same leaf showing ascocarps opening by 
stellate slits, magnified about 6 diameters. 

In the following three plates the figures are from camera lucida drawings and 
the magnification is indicated after each figure. 

Plate XIV 

Fig. 3. Section of leaf of Prunus avium showing the subepidermal acervulus 
bearing microconidia (spermatia ?) while still covered with macroconidia of Cylin- 
drosporium. X 250. 

Fig. 4. Pycnidium of Septoria pruni Ellis in leaf of Prunus amcricana, from 
"North American Fungi" II, 115 1. X 1,300. 

Fig. 5. Section of leaf of Prunus serotina showing subepidermal conidia bearing 
acervulus of Septoria cerasina Peck (= Cylindrosporium), from type material. 
X 250. 

Fig. 6. Ascocarp of Coccomyces hiemalis in leaf of Prunus avium, showing 
remains of epidermal cells above and below and Hgnified cells of host vascular bundle 
near center of ascocarp. X 250. ^ 

Fig. 7. Ascocarp of Coccomyces prunophorae. X 250. 

Fig. 8. Young ascocarp of Coccomyces hiemalis in leaf of Prunus avium 
showing portions of three ascogonial (?) coils and two trichogynes. The pseudo- 
parencyhmatous covering of the ascocarp is beginning to form and conidiophores 
(microconidiophores) are beginning to disintegrate above the center of the young 
fruit body. X 250. 

Fig. 9. Coiled ascogonial (?) branch from a slightly later stage in which the 
trichogyne is disintegrating. 

Fig. 10. Young asci and portions of ascogenous hyphae from a section of 
developing ascocarp of Coccomyces hiemalis. 

Fig. II. 'Old ascocarp of Coccomyces hiemalis producing "apothecial conidia." 
X 250. 

Plate XV 

Fig. 12. Section of leaf of Prunus virginiana showing entrance of germ tube 
of Cylindrosporium spore through a stoma and subsequent growth of mycelium. 
From artificial infection under bell jar four days after inoculation. X 450. 

Fig. 13. Early stage in the development of stroma and acervulus of Cylindro- 
sporium beneath the upper epidermis of same host, five days after inoculation. Two 
haustoria have entered an epidermal cell. X 450. 



172 BASCOMBE BRITT HIGGINS 

Fig. 14. Section of leaf of Prunus virginiana showing amphigenous nature of 
Cylindrosporium acervuli and the separation of the diseased spot. X 47. 

Fig. 15. Portion of section similar to fig. 14 more highly magnified showing 
the enlarged cells of separation layer and walls of ruptured cells. X 450. 

Fig. 16. Later stage, showing the suberized layer (the cell walls of which are 
represented by heavier lines) and the remains of the enlarged cells now dead. X 450. 

Fig. 17. Young haustoria, showing nuclei and vacuoles, in cells of bundle 
sheath in leaves of Prunus virginiana. 

Fig. 18. Haustoria in mesophyll cell of the same leaf surrounded by cellulose 
sheaths. 

Fig. 19. Developing ascocarp of Coccomyces lutescens in leaf of P. virginiana, 
showing the subepidermal position with remains of the gelatinized conidiophores and 
the early appearance of the paraphyses. X 250. 

Fig. 20. Mature ascocarp of same with asci and paraphyses. X 250. 

Fig. 21. Developing ascocarp of Coccomyces lutescens on Prunus serotina 
still covered by host epidermis. X 250. 

Fig. 22. Mature ascocarp of same. X 250. 

Plate XVI 

Fig. 23. Asci and single paraphysis of Coccomyces hiemalis from Prunus 
avium. X 450. 

Fig. 24. Ascospores of same more highly magnified. X 625. 

Fig. 25. Ascospores of same germinating in an agar culture five days old. 

Fig. 26. Asci and paraphyses of Coccomyces hiemalis from Prunus pennsyl- 
vanica, spores are mature in one on left. X 450. 

Fig. 27. Conidia (Cylindrosporium) of Coccomyces hiemalis on Prunus penn- 
sylvanica. X 450. 

Fig. 28. Asci and paraphysis of Coccomyces hiemalis on Prunus cerasus. 
The spores have been shed from one of the asci, and are scarcely mature in other. 

X 450. 

Fig. 29. Germinating conidia (Cylindrosporium) of Coccomyces hiemalis. 

X 450. 

Fig. 30. Microconidia (spermatia ?) and microconidiophores of Coccomyces 
hiemalis from P. avium. X 450. 

Fig. 31. Apothecial conidia of Coccomyces hiemalis on P. avium. X 450. 

Fig. 32. Same germinating after two days in water culture. 

Fig. 33. Asci and paraphyses of Coccomyces prunophorae from P. domestica. 

X 450. 

Fig. 34. Ascospores from same more highly magnified. X 650. 

Fig. 35. Apothecial conidia of Coccomyces prunophorae. X 450. 

Fig. 36. Conidia (Cylindrosporium) of Coccomyces prunophorae. X 450. 

Fig. 37. Ascus of Coccomyces lutescens from Prunus mahaleb. 

Fig. 38. Ascospores of same more highly magnified. X 625. 

Fig. 39. Microconidia and microconidiophores of Coccomyces lutescens. 

X 450. 



American Journal of Botany. 



Volume I, Plate XIII. 





HiGGiNs: Life History of Cylindrosposivm. 



\MERicAN Journal of Botany. 



Volume I, Plate XIV. 




HiGGiNs: Life Historv of Cylindrosporium. 



VoLUN'E 1, Plate XV. 



\MERiCAN Journal of Botany 




HIGOIXS: LIFE HISTORY OF CVLINDROSPORIL-M. 



American Journal of Botany. 



VOLUME I, Plate XVI. 




HiGGiNS: Life History of Cylindrosporium. 



CYLINDROSPORIUM ON STONE FRUITS 1 73 

Fig. 40. Ascus of Coccomyccs lutescens from P. virginiana. 

Fig. 41. Asci and paraphyses of Coccomyces lutescens from Prunus serotina. 

X 450. 

Fig. 42. Ascospores of same more highly magnified. X 625. 
Fig. 43. Apothecial conidia and conidiophores of Coccomyces lutescens on 
Prunus serotina. 

Fig. 44. Conidia (Cylindrosporium) of Coccomyces lutescens on Prunus 
serotina. 



